Chairs: Joan W. Bennett (JWB), Tulane University, USA and David W. Denning (DWD), University of Manchester, UK

Also attending: Michael Anderson (MA), University of Manchester, UK; Jonathan Arnold (JA), University of Georgia, USA; Jo-Anne van Burik, University of Minneapolis, USA; Yun Chang, NIAID, USA; Sandra Clifton, Washington University, USA; Dennis Dixon (DD), NIH, USA; Nancy Keller (NK), Texas A & M University, USA; Doris Kupfer, University of Oklahoma, USA; Jean-Paul Latgé (JPL), Institut Pasteur, France; Daniel Lawson (DL), Sanger Centre, UK; Victoria McGovern, Burroughs Wellcome (BW) Fund, USA; M Machida, National Institute of Bioscience and Human Technology, Japan; Michael Quail, Sanger Centre, UK; Geoff Turner (GT), University of Sheffield, UK; Ted White, Seattle Biomedical Research Institute, USA.
Apologies from: Rudolpho Arramayo, Bart Barrell, Andy Brass, Celia Caulcott, Philippe Glaser, Michael Gottlieb, June Kwon-Chung, Steve Oliver, Rolf Prade, Judy Rhodes, Bruce Roe, Marcel Ruiters, John Taylor.

In his opening statement, DWD introduced Af and its importance to the field of medical mycology. He stated that it was the most common filamentous fungal pathogen of humans and that the importance of sequencing its genome had been recognised by the NIH and the Wellcome Trust (WT) who had both listed it as an organism that needs to be sequenced.

The malaria project: D Lawson: introduced the Plasmodium falciparum (Pf) genome sequencing project, which has been going for 4 years with the sequencing being done during the past 3 years by the Sanger Centre, TIGR and Stanford. Funding is from BW, WT and the US gov. The consortium meet every 6 months. There are 14 chromosomes (chr) from size 0.7 to 3.5 Mb with a total genome of 30 Mb. Chr 2 and 3 were done as the pilot and have been finished. Pf has 82 % A+T in its genome with upto 95 % in intergenic regions with the result that gene islands are obtained which are difficult to link together. Some ways of mapping and joining together the contigs were discussed and other problems were mentioned such as predicting exons and determining telomeres and centromeres.
GT: A. nidulans (An) has an AT rich region near its centromeres and he mentioned that W Timberlake had been unable to walk across a centromere using cosmids.
DWD: when would Pf be finished? DL: in 2 years

The yeast project: Geoff Turner: because Steve Oliver was unable to attend and inform the meeting about yeast (S. cerevisiae) genomics, GT summarised a talk given by Bernard Dujon 1½ years ago. Main points were that in 40 years of yeast genetics, only ¼ of the genes had been studied; the EU funded project (EUROFAN) involving 138 labs is determining the function of orphan genes by systematic disruption and phenotypic analysis. Yeast was contrasted with A. nidulans: while 30 bp of flanking regions can be used for homologous recombination in yeast, at least 1 kb is required with An.

The Aspergillus nidulans chromosome IV project: Nancy Keller: described the project to sequence the smallest chromosome, IV (2 Mb) of An using the minimum tile (145) of cosmids generated by JA's group. The project was initiated by Tom Adams and Rolf Prade. NK took over project when T Adams moved to Cereon. All of the chr IV cosmids have been end-sequenced and complete sequencing of cosmids was started in Jan 99. Because funding has been provided by companies, there is a 6 month delay in the release of the data. The An EST project done by Bruce Roe at University of Oklahoma has helped with determining gene structure and location. This EST project has generated 13,000 sequences representing 3,100 genes and has now stopped because the redundancy reached 75 %.

Genoscope and the French perspective: Jean-Paul Latgé: provided an overview of scientists working on Af in Europe and of the French publicly funded genome sequencing facility - Genoscope. Genoscope has a scientific council which decides on which projects are to be funded and consists of 11 members. Two fungal projects have been funded including Botrytis cinerea. Deadlines for regular projects are in Oct with start dates 6 months later. Project sizes should enable 5 or 10 Mb of sequencing. JPL stated that Axel Brakhage had been informed by the German government that there was no money for genome sequencing.

Japanese efforts on fungal genomics: M Machida: provided an overview of Aspergillus projects from the Japanese perspective. The Fungal Genome Project of Japan was formed in 1997 and consists of 10 people from universities, industry and national institutes. One cosmid from chr VIII of An has been sequenced and the data will be released in July 1999. An EST project for A. oryzae has been set up. The FG project have been unsuccessful in getting funding from the government.

The A. fumigatus pilot project: Michael Anderson and Michael Quail: provided an overview of the pilot project to sequence Af funded by the Wellcome Trust. Additional points that were made included the fact that the strain would be sent out to anyone who wanted it, that the partial digestion of the chromosomal DNA would be done using varying ratios of EcoRI to EcoRI methylase and that all the BAC clones (at least 3000 - 10 x coverage) would be end-sequenced.

Analysis, annotation and curatorship: David Denning (in place of Andy Brass): overviewed 3 levels of annotation - 1) identification of ORFs and repetitive sequences; 2) automated computer analyses including Blast and protein motif searches and 3) human (biologist) assignment of putative function. He proposed that Manchester be responsible for the annotation and act as curator of the database, which met with general approval. He raised concerns about how to involve the diverse Aspergillus community and suggested that the relevant experts in the various fields validate the initial assignments of putative gene function.

General discussion:
Karyotype: the need for a decent karyotype and an investigation into the level of strain variation was stressed. MA showed a CHEF gel photograph which indicated that there was variation between strains.

JLP stated that a karyotype had been published which showed 5 bands. A karyotype would assist with the mapping of the BAC clones.
To be done: Jo-Anne van Burik and JWB will investigate the possibility of obtaining funding to carry out a karyotyping project.

Collaborations: the point was made that An researchers would not be interested in doing some of the sequencing, but that they might be interested in helping with annotation or in doing some of the biology. Co-operation could also be extended from the current projects being funded by the EU.

The mechanics of the project: It was pointed out that although there was access to Af cosmid libraries, it was essential that a cosmid library be made from the same strain as is being used in the pilot project. This was especially so if there is the possibility of variation in genome structure between strains of Af. The point was also made that the cloning of telomeres would probably have to be done specifically.
There was debate about how to get the BAC clones sequenced - do we use only a few large sequencing centres or do we send out one or two clones to any group that asks for them? Do we consider the reputation of the centre? How do we validate the sequences? How much does each base cost? Once a policy has been decided, the Sanger centre would act as co-ordinators since they have the facilities for storage of the clones.
To be done: Sequencing centres need to be identified and approached. A detailed sequencing plan involving suitably sized modules for funding purposes, needs to be put together and approved.

Cost: the total project was estimated to cost at least $15-20 million for the sequencing and first pass annotation, assuming a minimum cost of $ 0.50 per base and excellent tiling. Applications to the NIH for the money which has been earmarked for the sequencing of pathogen genomes, would be for sequencing and first pass annotation. Two of these projects will be funded each year, but DD stated that final details have still to be decided. NK stated that the US Department of Agriculture has money for microbial genomes. The National Science Foundation however will not provide money for pathogens. It was thought likely that more money would not be available from the WT until the end of the pilot project (01/2001). Money should be available from the French government through Genoscope and DWD thought it should be possible to obtain money from the EU through the Framework V programme. Some money should be available from the NIH for standard hypothesis driven projects.
To be done: various participants of this meeting including DWD, JPL, NK and MM need to explore the possible sources of funding so that all the details are ready for applications to be submitted when the BAC library has been made and mapped. DWD is specifically going to address accessing funding from the Japanese government.

Annotation: there was some debate about what level of annotation would be required and DL made a strong case for the level currently being done at the Sanger centre using the AceDB database set-up.  First pass annotation involves answering the question - what is the gene? and requires an annotator who is a biologist to provide an subjective angle on the automated computer analyses. JPL stated that he considered the level provided by the Sanger centre to be good and DL made the point that there would be the need for continued curation of the database after the end of the sequencing period. It was stated that the annotation problem is as big as the sequencing problem. DWD proposed using the fungal community to help with annotation. The data from the Af EST project currently underway in DWD's lab with sequencing being done in Roe's lab (U of Oklahoma) should help with the location of some of the ORFs and with the positioning of exons. JA stated that he had applied to the NSF and NIH for money for 7 annotators to work on Neurospora crassa and will receive help from Proteome. Proteome employs 25 PhDs at the moment and JPL stated that they are already doing the annotation for Candida albicans genes. Because of this, it was suggested that we should seek a possible collaboration with Proteome and that they would probably welcome such an approach. It was suggested that the database should start off simply, but we should have a perceived vision to help with funding applications. This annotated database was not considered by some participants to have a commercial value since industry employs their own bioinformatics teams. However, a detailed annotation would add enormous value to the usefulness of the sequence data for the scientific community, in addition to forming new links between members of the Aspergillus communities. The point was made that, since Af is not a model organism, then the level of annotation need not be high.
To be done: DWD will canvass opinion about what annotation would be useful using examples posted on the Aspergillus web site. A format should be decided on for the next meeting.

Nomenclature: a strong case was made that the naming of the genes must follow a formalised convention. JWB and L Lasure have already written a paper on classical gene notations and this could be used as a starting point. Options include using the An convention or the yeast convention. It was stated that with the An EST project, no nomenclature convention had been adopted and that the ESTs were simply coded. DL stated that for the 3 Trypanosome species which are being sequenced, a meeting had been held, the agreed convention was checked with those people not at the conference and submitted as a short paper to a journal. Next there was some debate about whether to use a species prefix in a similar manner to that currently used by EMBL (e.g. Afu). DL stated that the Trypanosome meeting decided against using a species prefix.
To be done: either: to arrange a forum at a suitable meeting or to canvass people by electronic means with the various alternatives.

Functional genomics: JPL stated that working with Af will be a lot more time consuming than yeast with gene analysis at the rate of 1 gene/ 1 person / 1 year (to make the mutant and analyse its phenotype; to synthesize recombinant protein and study its function). Functional complementation in yeast was proposed as a possibility using the EUROFAN deletants and also in An using those genetic mutants without an identified gene.

General points: profile of this project needs to be raised at relevant meetings. It was considered likely by some participants that the An sequence would follow since chr IV was being done. Also, it was felt that An researchers would be willing to lend their expertise and experience, although it was felt unlikely that they would want to work on Af because it is a human pathogen and has no sexual cycle. The project would be co-ordinated in Manchester and details posted on the Aspergillus Web Site. The next co-ordination meeting would be held at the Sanger centre tentatively in Jan 2000.

Workshop Photo One

Workshop Photo Two

30th June 1999 - updated June 16th 2000
Michael Anderson
David Denning
Joan Bennett